CONFIDENTIAL. For Internal Use Only. © 2010 Life
Rafal P. Witek, Kirsten B. Amaral, Manda A. Edwards, Adam M.
Farmer, Kimberly M. Freeman, Erica Deibert, Tania Bembridge,
Jonathan P. Jackson, Cornelia Smith, Jasminder Sahi, Stephen S.
Ferguson, Mark J. Powers.
ADMETox/ Life Technologies, Durham NC USA
Introduction: Kupffer cells are activated liver resident
macrophages that play an important role
in liver immunity and in the modulation of xenobiotic metabolism
during liver inflammation.
These cells suppress the expression of multiple cytochrome P450
enzymes through the
exchange of soluble factors, following activation with either
lipopolysaccharide (LPS) or other
pro-inflammatory cytokines. Since those interactions lead to
severe pharmacological and
toxicological consequences, there is an evident need to develop
a commercially available model
mimicking the liver inflammatory state that could be used for in
vitro drug metabolism and
discovery ADME/Tox applications. We use a liver co-culture
system consisting of plated primary
hepatocytes and Kupffer cells in a physiologic 2:1 ratio, which
is relevant to inflammation, and
compare these to monocultures of Kupffer cells or hepatocytes.
Material and Methods: Primary
rat Kupffer cells and hepatocytes were isolated using a modified
two step collagenase perfusion.
Purity of isolated Kupffer cells was determined by culturing
cells for 2 days and analyzing them
by immunostaining for ED1 and ED2 expression. To create
inflammatory co-cultures, Kupffer
cells were seeded at a density approximating half that of
hepatocytes and cultured for 48hr prior
to all experiments. Kupffer cell activation was induced by
treating hepatocyte cultures, Kupffer
cell cultures, or co-cultures with LPS (1µg/mL) or IL2
(200ng/mL) for a durations of 24, 48 and
72hrs. Following treatment, all cultures were analyzed for
morphology, cytokine production by
ELISA, and P450 mRNA expression (CYP3A and CYP1A2). Results:
Isolated Kupffer cells
expressed ED1 at 88.7% (±1.05) and ED2 at 75.5% (±5.4) after 2
days in culture. Following 6 days in culture, expression of ED1
remained at 85.6% (±1.9) and ED2 expression significantly increased
to 93.5% (±0.4) suggesting Kupffer cell activation. As expected,
following plating and activation, primary Kupffer cells
significantly increased production of pro-inflammatory
IL-6 and TNFa in co-cultures and Kupffer cell monocultures.
However, at 72hr after treatment
with either LPS or IL2, only co-cultures showed upregulated
expression of IL6 by nearly 2-fold
compared with monocultures of Kupffer cells. This suggests
auto-assembly of cellular
interactions between both types of cells. After 72hr treatment,
LPS and corresponding IL-6 up-
regulation correlated with approximately 20% down-regulation of
CYP3A and CYP1A2
expression. IL2 treatment correlated with approximately 80%
down-regulation of both enzymes.
Conclusion: Our data suggests that Kupffer cell and hepatocyte
co-cultures can self-assemble
within 72hr of treatment with pro-inflammatory cytokines or LPS,
and can function by effectively
modulating P450 expression in co-cultured hepatocytes. As such,
co-culture of primary
hepatocytes and Kupffer cells mimicking liver inflammatory state
offer a powerful in vitro
ADME/Tox tool to assess the effects of xenobiotics on drug
metabolizing enzymes and more
closely model more complex hepatic cell biology in vitro.
The largest solid organ in the human body, the liver is
responsible for diversity of function with
most important being metabolism, detoxification and protein
synthesis. Most of those functions take place within hepatocytes
which represent a major cell type of the hepatic parenchyma;
also contains a large proportion of non-parenchymal cells (NPCs)
which include liver sinusoidal endothelial cells, hepatic stellate
(ito) cells, cholangiocytes and Kupffer cells. In general, NPCs
provide physical and biochemical structure to the liver. Out of all
NPCs, Kupffer cells, which are the liver resident
macrophages, play an important role in liver physiology and
homeostasis by participating in the acute and chronic responses of
the liver to toxic compounds during liver inflammation.
In the liver, Kupffer cells are located on the sinusoidal side
of hepatic parenchyma and use their stellate like cytoplasmic
extensions for direct cell-to-cell contact with hepatocytes. This
essential for proper modulation and the development of a
fulminant hepatic inflammatory response. In addition to cell
contact, activation of Kupffer cells results in the release of a
variety of inflammatory
cytokines and growth control mediators that suppress the
expression of multiple cytochrome P450 enzymes. Two of those
factors, Interleukin 6 (IL6) and Tumor Necrosis Factor-a (TNFa) are
induce the synthesis of acute phase proteins and suppress
CYP1A2, CYP2C19 and CYP3A activities. Those three P450 enzymes
represent the main group of metabolic enzymes involved in
detoxification of xenobiotic substances.
-Kupffer cells secrete potent mediators of the inflammatory
response that control liver
inflammation and hepatocyte metabolic rates through direct
interactions with phase I and phase II enzymes.-Kupffer cells can
be used to create co-culture liver inflammatory system when plated
with hepatocytes-Isolated primary rat Kupffer cells are >90%
pure as determined by immune stain for ED1 and
ED2.-Plated Kupffer cells culture activate as determined by
increase expression of resident
macrophage marker ED2.-Kupffer cells and hepatocytes
self-assembly in culture-IL2 or LPS activated Kupffer cells
modulate CYP3A and CYP1A2 activities
REFERENCES Hoebe KH, Witkamp RF, Fink-Gremmels J, Van Miert AS,
Monshouwer M. Direct cell-to-cell
contact between Kupffer cells and hepatocytes augments
endotoxin-induced hepatic injury. Am J Physiol Gastrointest Liver
Physiol. 2001 Apr;280(4):G720-8.
Sunman JA, Hawke RL, LeCluyse EL, Kashuba AD. Kupffer
cell-mediated IL-2 suppression of
CYP3A activity in human hepatocytes. Drug Metab Dispos. 2004
Roberts RA, Ganey PE, Ju C, Kamendulis LM, Rusyn I, Klaunig JE.
Role of the Kupffer cell in
mediating hepatic toxicity and carcinogenesis. Toxicol Sci. 2007
Mar;96(1):2-15. Epub 2006 Nov 22.
Co-culture of Hepatocytes and Kupffer
Cells as a Model for Liver Inflammation.
MATERIALS AND METHODS
Rat Kupffer cell isolation: Primary rat Kupffer cells were
isolated using proprietary enzymatic digestion method developed at
Life Technologies. Using this technique, Kupffer cells can be
isolated at viabilities
higher than 98% and purities of approximately 90% or higher as
determined by immune stain for ED1 (CD68) and ED2 (CD163) markers.
Contaminating cells represent small populations of endothelial
~3%, fibroblasts ~2%, hepatocytes 1%, and Kupffer cells that did
not stain sufficiently to be counted). Isolated Kupffer cells
attach within 15-30min and display macrophage morphological
characteristics within 24hr. They become activated and ready for
experiments within 48hr after plating. Kupffer cells are
cultured in supplemented WEM.
Rat Hepatocyte isolation: Primary rat hepatocytes are isolated
using two step collagenase perfusion. Cells with viability of at
least 85%, as determined by trypan blue exclusion, are used in
Supplemented WEM was used during culture and changed every 24
Co-Cultures: Hepatocytes and Kupffer cells were co-cultured in
24-well dishes at ratios of 1:2 of Kupffer
cells/hepatocytes to approximate physiological inflammatory
liver state. Additional 1:4, 1:8 and 1:16 ratios were used to
determine linearity of inflammatory response. Supplemented WEM was
used in co-
culture and changed every 24 h.
Data Analysis: To activate inflammatory response in Kupffer
cells, co-cultures were treated with either LPS (1ug/ml) or IL2
(200ng/ml). Response was analyzed at 24, 48 or 72hr for IL6 and
TNFa production using ELISA assays. For those assays, media was
collected from each well at predetermined time
points and stored at -80oC until analysis. To perform qRT-PCR,
at terminal time point, cells were washed with HBSS and total RNA
Following cDNA synthesis, TaqMan analysis was performed using
probes for rat CYP1A2 and CYP3A23
Figure 1. Morphological characterization of primary rat Kupffer
cells. A. At 2hr after plating, rat Kupffer cells display fried egg
like morphology of immature macrophages; B. Within
24hr after plating, rat Kupffer cells expand and activate to
their mature macrophage-like phenotype; C-D. After 48hr to 96hr
after plating and thereafter, Kupffer cells become fully activated
to their stellate-like phenotype that is characteristic of liver
resident macrophages and
further validated by ED2 (CD163) immune stain.
Figure 3. Primary rat Kupffer cell co-cultures with hepatocytes.
A. Representative image of
primary rat monoculture of hepatocytes plated for 48hr
displaying characteristics of epithelial monolayer; B.
Representative image of inflammatory co-culture representing the
1:2 ratio of Kupffer cells to hepatocytes. Note stellate like
morphology of Kupffer cells and their proximity to
Figure 4. Progressive media accumulation of IL6 and TNFa in
co-cultures of Kupffer cells
and hepatocytes. Cells were plated at different ratios
corresponding to those of normal (1:16) and inflamed (1:8 – 1:2)
liver at 24hr after treatment with 1ug/ml of LPS. .
Figure 2. Activation of primary rat Kupffer cells in culture.
A1-A3. Immune characterization
of Kupffer cells at 48hr after plating for presence of ED1 (A1)
and ED2 (A2) macrophage markers reveals their expression at 88.7%
and 75.5%, respectively (A3). B1-B3. Following
activation at 6 days after plating, Kupffer cells express ED1
(B1) at 85.6% and ED2 (B2) at 93.5% (B3).
Figure 5. IL6 production in Kupffer cells after LPS and IL2
stimulation for 24, 48 and
72hr. As expected, following activation with LPS treatment, IL6
production is significantly up-regulated at 24hr and then it
progressively decreases suggesting desensitization of Kupffer cells
to chronic stimulation with LPS.
Figure 6. IL6 production in Kupffer cells and hepatocyte
co-cultures after LPS and IL2
stimulation for 24, 48 and 72hr. Note that IL6 is significantly
up-regulated in co-cultures at
all time points of 24, 48 and 72hr. This suggests cellular self
assembly between Kupffer cells and hepatocytes that synergistically
allows those cells to function together during resolution of
Figure 7. Modulation of CYP3A2 and CYP1A2 enzyme activities in
co-cultures after LPS
and IL2 stimulation for 72hr. Note near 80% decrease of CYP3A23
and CYP1A2 after IL2 treatment.
For Research Use Only. Not intended for any animal or human
therapeutic or diagnostic use